Reversal of heavy arterial calcification in a rat model of chronic kidney disease using targeted ethylene diamine tetraacetic acid-loaded albumin nanoparticles.

IF 2.1 3区 医学 Q3 CARDIAC & CARDIOVASCULAR SYSTEMS
Cardiovascular diagnosis and therapy Pub Date : 2024-08-31 Epub Date: 2024-08-23 DOI:10.21037/cdt-24-17
Fatema Tuj Zohora, Shivani Arora, Alyssa Swiss, Naren Vyavahare
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引用次数: 0

Abstract

Background: Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy.

Methods: Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification in vitro, and then they were treated with EDTA. Human calcified femoral arteries were also treated ex vivo with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification.

Results: EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs vs. 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 vs. 29.815±24.169 mm3, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of BMP2 and RUNX2 (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (BMP2, RUNX2) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show ex vivo reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 vs. day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy.

Conclusions: This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.

使用靶向乙二胺四乙酸负载白蛋白纳米粒子逆转慢性肾病大鼠模型中的重度动脉钙化。
背景:血管壁内侧层的弹性蛋白降解和严重钙化,即内侧动脉钙化(MAC),是老龄人口和代谢性疾病(如糖尿病和慢性肾病)患者的典型症状。我们以前曾报道过,通过在纳米粒子上标记可识别明确受损弹性蛋白的弹性蛋白抗体,可将乙二胺四乙酸(EDTA)输送到钙化部位,这种全身性疗法可清除 CKD 啮齿动物模型钙化动脉中的钙沉积灶。本研究旨在测试纳米粒子疗法能否逆转 CKD 患者动脉树和肾脏中的严重钙化:方法:将 30 只体重约 300 克的健康雄性 Sprague-Dawley 大鼠置于腺嘌呤饮食中,连续 21 天诱导肾衰竭,然后连续 4 天每天注射维生素 D3 (VitD3),导致整个心血管系统和肾脏严重钙化。为了证实纳米粒子对钙化区域的靶向性,使用了装载 DiR 染料和弹性蛋白抗体共轭的白蛋白纳米粒子。将大鼠分为两组,从最后一次服用 VitD3 的第 7 天开始进行靶向清除钙化。实验组每两周静脉注射一次抗弹性蛋白抗体共轭 EDTA 负载人血清白蛋白纳米粒子(EDTA-HSA-El-Ab NPs),而假对照组则注射空白纳米粒子(Blank-HSA-El-Ab NPs)(共注射 5 次)。显微计算机断层扫描(microCT)用于分析钙化程度。对骨形态发生蛋白 2 (BMP2)、runt 相关转录因子 2 (RUNX2) 和组织非特异性碱性磷酸酶 (TNAP) 等成骨标志物进行了逆转录聚合酶链反应 (RT-PCR) 和免疫组化研究。为了进行比较,先用高磷酸盐处理健康大鼠的主动脉环器官培养物以诱导体外钙化,然后再用乙二胺四乙酸处理它们。还在体外用 EDTA-HSA-EL-Ab NPs 处理人体钙化的股动脉,以检验纳米颗粒是否能去除重度钙化:结果:根据钙元素分析(Blank-HSA-El-Ab NPs 组每毫克干主动脉含 124.161±34.410 微克钙,EDTA-HSA-El-Ab NPs 组每毫克干主动脉含 100.520±19.131 微克钙,P=0.04)和 microCT(物体体积,129.001±37.785 vs. 29.815±24.169 mm3,P=0.0005),EDTA 负载纳米粒子专门针对降解的弹性蛋白,逆转了动脉中现有的重矿物质沉积。在逆转主动脉钙化的同时,BMP2 和 RUNX2 的骨相关 mRNA 表达也显著减少(P=0.01)。免疫组化研究证实了 RT-PCR 的结果,显示血管壁中的 BMP2 和 RUNX2 染色减少。大鼠主动脉环培养研究也显示了类似的结果,用 EDTA 逆转钙化后,成骨基因(BMP2、RUNX2)和蛋白质(BMP2、RUNX2、TNAP)受到抑制(P=0.001)。我们还通过显微 CT 显示,EDTA 纳米粒子疗法可在体外逆转人体股动脉钙化(钙强度:未治疗时,57.721±28.551 vs. 治疗第 6 天,5.441±3.615,P=0.01):结论:这是第一项通过静脉注射 EDTA 靶向疗法清除严重钙化动脉中钙的研究。这种疗法还能逆转动脉组织中血管平滑肌细胞的成骨细胞转化和凋亡,从而有可能为合适的组织修复创造环境。
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来源期刊
Cardiovascular diagnosis and therapy
Cardiovascular diagnosis and therapy Medicine-Cardiology and Cardiovascular Medicine
CiteScore
4.90
自引率
4.20%
发文量
45
期刊介绍: The journal ''Cardiovascular Diagnosis and Therapy'' (Print ISSN: 2223-3652; Online ISSN: 2223-3660) accepts basic and clinical science submissions related to Cardiovascular Medicine and Surgery. The mission of the journal is the rapid exchange of scientific information between clinicians and scientists worldwide. To reach this goal, the journal will focus on novel media, using a web-based, digital format in addition to traditional print-version. This includes on-line submission, review, publication, and distribution. The digital format will also allow submission of extensive supporting visual material, both images and video. The website www.thecdt.org will serve as the central hub and also allow posting of comments and on-line discussion. The web-site of the journal will be linked to a number of international web-sites (e.g. www.dxy.cn), which will significantly expand the distribution of its contents.
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